| In recent years,the emergence of "super bacteria" has brought great threat to human life safety.The multidrug-resistant Acinetobacter baumannii has a resistance rate of more than 90% in the ICU,and there are few antibiotics that are effective in clinical practice.Therefore,phage therapy has gradually attracted people’s attention.However,in the face of this "super bacteria ",even phage will face the problem of producing resistant strains,so the combination of drugs is a new trend.The combination of phage and antibiotics not only produces better antibacterial effects,but also inhibits the production of respective resistant bacteria and produces synergistic effects.At the same time,new phage can be continuously selected to produce new combinations.The aim of the study was to isolate the lytic phage with multi-drug resistant Acinetobacter baumannii,and then cooperate with antibiotics for planktonic bacteria and biofilms to select for antibacterial combinations with obvious synergistic effects.This experiment is mainly divided into the following three parts:Part Ⅰ: A lysis phage was obtained by double-layer plate separation and purification,and named M1;its plaque was about 3 mm in diameter,the center was bright and transparent and there was a halo around it;under electron microscope,the head had a regular hexahedral structure with a diameter of about 50 mm.With a non-shrinkable short tail,belonging to the short tail phage;the optimal multiplicity of infection is 0.001;the incubation period is less than 10 min,the lysis period is about 55 min,and the lysis amount is about 300 PFU/infected cell;Phages are highly stable to temperature and pH;there was no significant difference in the antibacterial effect of the phage M1 with different multiple infection(P>0.05).Part Ⅱ: Optimal multiplicity of infection of phage M1 and 8 antibiotics were combined to affect on planktonic bacteria,and the synergistic effect was detected by monitoring bacterial and phage concentrations.The results showed that phage M1 combined with MIC rifampicin and 1/2MIC meropenem had a synergistic effect,with 1/4 MIC of minocycline,with strong synergistic effect;When phage is used in combination with sensitive antibiotics,the choice of antibiotic concentration and the order of addition have an effect on synergy.Part Ⅲ: The combination of phage M1 and antibiotics was applied to the biofilm to observe if there was synergy.The removal of biofilm was evaluated by crystal violet staining and viable count method.The results showed that phage M1 could clear 98% of the biofilm at 4 h,but with the prolongation of time(12 h),the clearing ability of the biofilm gradually weakened until it disappeared;phage M1 and 1/2 MIC of rifampicin Combined action on the biofilm for 12 h can produce a significant synergistic effect.Conclusion: A phage M1 capable of lysing multi-drug resistant Acinetobacter baumannii was isolated and obtained with stable biological characteristics,short incubation period and strong in vitro cleavage ability;Phage M1 and rifampicin When meropenem and minocycline are combined to act on planktonic bacteria,they can significantly inhibit the production of each resistant bacteria and produce a synergistic effect,and the strength of synergy is related to the concentration of antibiotics and the order of application;Phage M1 can clear 98% of the biofilm within 4 hours,and it also has a synergistic effect when it is applied to the biofilm in combination with rifampicin. |
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